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Xanthomonas perforans and X. euvesicatoria are the causal agents of bacterial spot disease of tomato and pepper, endemic to the Southeastern United States. Although very closely related, the two bacterial species differ in host specificity, where X. perforans is the dominant pathogen of tomato and X. euvesicatoria that of pepper. This is in part due to the activity of avirulence proteins that are secreted by X. perforans strains and elicit effector-triggered immunity in pepper leaves, thereby restricting pathogen growth. In recent years, the emergence of several pepper-pathogenic X. perforans lineages has revealed variability within the bacterial species to multiply and cause disease in pepper, even in the absence of avirulence gene activity. Here, we investigated the basal evolutionary processes underlying the host range of this species using multiple genome-wide association analyses. Surprisingly, we identified two novel gene candidates that were significantly associated with pepper-pathogenic X. perforans and X. euvesicatoria. Both candidates were predicted to be involved in the transport/acquisition of nutrients common to the plant cell wall or apoplast and included a TonB-dependent receptor, which was disrupted through independent mutations within the X. perforans lineage. The other included a symporter of protons/glutamate, gltP, enriched with pepper-associated mutations near the promoter and start codon of the gene. Functional analysis of these candidates revealed that only the TonB-dependent receptor had a minor effect on the symptom development and growth of X. perforans in pepper leaves, indicating that pathogenicity to this host might have evolved independently within the bacterial species and is likely a complex, multigenic trait.

For most of the 20th century, Xanthomonas euvesicatoria was the only known bacterium associated with bacterial spot of tomato in Florida. X. perforans quickly replaced X. euvesicatoria, mainly because of production of three bacteriocins (BCNs) against X. euvesicatoria; however, X. perforans outcompeted X. euvesicatoria even when the three known BCNs were deleted. Surprisingly, we observed antimicrobial activity against X. euvesicatoria in the BCN triple mutant when the triple mutant was grown in Petri plates containing multiple spots but not in Petri plates containing only one spot. We determined that changes in the headspace composition (i.e., volatiles) rather than a diffusible signal in the agar were required for induction of the antimicrobial activity. Other Xanthomonas species also produced volatile-induced antimicrobial compounds against X. euvesicatoria and elicited antimicrobial activity by X. perforans. A wide range of plant pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis, Pantoea stewartii, and Pseudomonas cichorii, also elicited antimicrobial activity by X. perforans when multiple spots of the species were present. To identify potential antimicrobial compounds, we performed liquid chromatography with high-resolution mass spectrometry of the agar surrounding the spot in the high cell density Petri plates where the antimicrobial activity was present compared with agar surrounding the spot in Petri plates with one spot where antimicrobial activity was not observed. Among the compounds identified in the zone of inhibition were N-butanoyl-L-homoserine lactone and N-(3-hydroxy-butanoyl)-homoserine lactone, which are known quorum-sensing metabolites in other bacteria.

Although cultivars possessing recessive resistance alleles provide effective control of bacterial spot of pepper (Capsicum annuum), the deployed resistance gene, bs5, is ineffective against Xanthomonas gardneri, one of the pathogenic species. Resistance against X. gardneri was identified in C. annuum accession PI 163192, and this study sought to characterize this novel resistance and to map the resistance gene(s) to the pepper genome. We crossed PI 163192 with the susceptible cultivar Early Calwonder (ECW) to develop resistant near-isogenic lines (NILs) of ECW, designated ECW80R. The novel resistance in ECW80R was determined to be quantitative, recessively inherited, and non-hypersensitive-response causing, and inhibits lesion expansion and chlorosis. Presence of the resistance in NILs decreased the in planta bacterial population by ninefold compared with ECW. Bulked segregant analysis of resistant and susceptible individuals from an F2 population using whole genome single nucleotide polymorphisms identified a major resistance locus within an approximate 6-Mbp interval on the subtelomeric region of chromosome 11. We developed markers spanning this region and used these to genotype backcross F2 populations, which further delimited the resistance locus within a 2.3-Mbp interval. The novel resistance locus has been designated bs8. ECW80R and the linked markers developed in this study should prove useful for breeders seeking to advance this resistance into commercially relevant germplasm and for pyramiding bs8 with other resistance alleles such as bs5 and bs6. The allele bs8 will help prolong the durability of bacterial spot resistance in pepper and improve resistance to multiple species of Xanthomonas.

Bacterial spot is an economically significant disease in tomato and pepper-producing countries globally. We report the whole genome sequence of 11 Xanthomonas strains associated with bacterial spot disease on pepper, tomato, and eggplant in the Southeastern Anatolia Region, Turkey. This genomic information can be used as a reference to study the genetic diversity of these species and contribute to illuminating pathogen evolution with respect to host specificity.

Five bacterial strains were isolated from symptomatic leaves of Achillea millefolium, Delphinium sp. and Hydrangea sp. in California. Colonies isolated on King’s medium B (KMB) appeared white, mucoid and round, similar to Pseudomonas species. Phylogenetic analyses based on 16S rRNA, rpoB, rpoD and gyrB genes placed the bacteria into three distinct groups within Pseudomonas that were most closely related to Pseudomonas viridiflava , Pseudomonas cichorii or Pseudomonas caspiana . To further characterize the strains, phenotypic analyses and the following tests were performed: fatty acid methyl ester composition, LOPAT, fluorescence on KMB, Biolog assay, and transmission electron microscopy. Finally, whole genome sequencing of the strains was conducted, and the sequences were compared with reference genomes of Pseudomonas species based on average nucleotide identity (ANI). The first group, which consists of three strains isolated from delphinium, hydrangea and achillea, had 95.6–96.9 % pairwise ANI between each other; the second group consists of two strains isolated from delphinium that had 100 % pairwise ANI. Although comparisons of the two groups with publicly available genomes revealed closest relationships with P. viridiflava (91.6 %), P. caspiana (88.3 %) and P. asturiensis (86.7 %), ANI values were less than 95 % compared to all validly published pseudomonads. Combining genomic and phenotypic data, we conclude that these strains represent two new species and the names proposed are Pseudomonas quasicaspiana sp. nov. (type strain DSMZ 11 30 42T=LMG 32 434T) for the strains isolated from delphinium, achillea and hydrangea and Pseudomonas californiensis sp. nov. (DSMZ 11 30 43T=LMG 32 432T) for the two strains isolated from delphinium. The specific epithets quasicaspiana and californiensis were selected based on the close phylogenetic relationship of strains with P. caspiana and on the geographic location of isolation, respectively.

Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregant populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6 of pepper. We also identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. Mapping of bs5 and bs6 will facilitate their use in breeding programs through marker-assisted selection and is also a crucial step towards understanding the mechanisms of resistance.Key messageTwo recessive bacterial spot resistance genes were mapped in the pepper genome, which will facilitate their advancement in commercial pepper for management of all races of Xanthomonas euvesicatoria.

For most of the 20th century

The pathogen that causes stem gall in Loropetalum chinense was first identified in Florida and Alabama in 2018 and named Pseudomonas amygdali pv. loropetali. We report the genome sequence of the pathotype strain of this pathogen, Pseudomonas amygdali pv. loropetali DSM105780 PT.

Before 1991, Xanthomonas euvesicatoria was the causal agent of bacterial spot of tomato in Florida but was quickly replaced by X. perforans. The X. perforans population has changed in genotype and phenotype despite lack of a clear selection pressure. To determine the current Xanthomonas population in Florida, we collected 585 Xanthomonas strains from 70 tomato fields, representing 22 farms across eight counties, in the Florida tomato production region. Strains were isolated from 23 cultivars across eight seed producers and were associated with eight transplant facilities during the fall 2017 season. Our collection was phenotypically and genotypically characterized. Only X. perforans was identified, and all strains except one (99.8%) were tolerant to copper sulfate and 25% of strains were resistant to streptomycin sulfate. Most of the strains (99.3%) that were resistant to streptomycin sulfate were sequence type 1. The X. perforans population consisted of tomato races 3 (8%) and 4 (92%) and all three previously reported sequence types, ranging from 22 to 46% frequency. Approximately half of all strains, none of which were sequence type 2, produced bacteriocins against X. euvesicatoria. Effector profiles were highly variable among strains, which could impact the strains’ host range. The effector xopJ4, which was previously thought to be conserved in X. perforans tomato pathogens, was absent in 19 strains. Nonmetric multidimensional scaling and network analyses show how strains and strain traits were associated with production system variables, including anonymized farms and transplant facilities. These analyses show that the composition of the Florida X. perforans population is diverse and complex.

The deferred antagonism technique has been utilized for several decades for detecting antibiosis activity. Most protocols require the elimination of antibiotic-producing cells by exposing them to chloroform vapour, UV radiation or filter sterilizing the filtrate steps that require additional time and expense to complete. We provide a modified approach to current soft agar overlay practices, which involves addition of antibiotics to the soft agar overlay to inhibit growth of the producer but not the indicator strain. This technique can be used to reproducibly and efficiently screen for antibiotic production with ease. We demonstrate the effectiveness of this technique with three bacterial systems: inhibition of the bacterial spot of tomato pathogen, Xanthomonas euvesicatoria, by its pathogenic competitor Xanthomonas perforans; and inhibition of the fire blight pathogen, Erwinia amylovora, by Pantoea vagans C9-1 or Pseudomonas fluorescens A506. Significance and Impact of the Study Deferred antagonism assays are used commonly to observe antibiotic production by micro-organisms. Killing or removing the producer cells prior to introduction of the indicator strain is a standard practice but requires additional time and special handling procedures. We evaluated a modification of the assay, where the overlay medium is amended with an antibiotic to which the indicator strain is resistant and the producer strain is sensitive. This modification obviates extra steps to kill the producer strain prior to overlaying with the indicator strain and provides a rapid, consistent and cost-effective method to detect antibiosis.

Bacterial spot disease was first reported from South Africa by Ethel M. Doidge in 1920. In the ensuing century after the initial discovery, the pathogen has gained global attention in plant pathology research, providing insights into host–pathogen interactions, pathogen evolution, and effector discovery, such as the first discovery of transcription activation-like effectors, among many others. Four distinct genetic groups, includingXanthomonas euvesicatoria(proposed name:X. euvesicatoriapv.euvesicatoria),Xanthomonas perforans(proposed name:X. euvesicatoriapv.perforans),Xanthomonas gardneri(proposed name:Xanthomonas hortorumpv.gardneri), andXanthomonas vesicatoria, are known to cause bacterial spot disease. Recently, a new race of a bacterial spot pathogen, race T5, which is a product of recombination between at least twoXanthomonasspecies, was reported in Nigeria. In this review, our focus is on the progress made on the African continent,vis-à-visprogress made in the global bacterial spot research community to provide a body of information useful for researchers in understanding the diversity, evolutionary changes, and management of the disease in Africa.

Field trials were conducted at two locations in Florida to evaluate transgenic tomato expressing the ELONGATION FACTOR TU RECEPTOR (EFR) gene from Arabidopsis thaliana, the Bs2 gene from pepper, or both Bs2 and EFR (Bs2/EFR) for managing bacterial wilt caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans. Expression of EFR or Bs2/EFR in the susceptible genotype Fla. 8000 significantly reduced bacterial wilt incidence (50 to 100%) and increased total yield (57 to 114%) relative to lines expressing only Bs2 or the nontransformed Fla. 8000 control, although the marketable yield was not significantly affected. Following harvest, surviving symptomatic and nonsymptomatic plants were assessed for colonization by R. solanacearum. There were no significant differences in the population at the lower stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR or Bs2/EFR lines but viable bacterial populations were recovered from Bs2 and nontransformed control lines at 102to 105CFU/g of stem tissue. In growth-chamber experiments, the EFR transgenic tomato lines were found to be effective against seven different R. solanacearum strains isolated from the southeastern United States, indicating utility across the southeastern United States. In all of the bacterial spot trials, EFR and Bs2/EFR lines had significantly reduced disease severity (22 to 98%) compared with the Fla. 8000 control. The marketable and total yield of Bs2/EFR were significantly higher (43 to 170%) than Fla. 8000 control in three of four field trials. These results demonstrate for the first time the potential of using the EFR gene for field management of bacterial wilt and bacterial spot diseases of tomato.

Pseudomonas syringae pv. Tomato causes bacterial speck in tomato. We report the genome sequences of two P. syringae pv. Tomato strains, J4 and J6, that are genetically closely related, with >99.9 average nucleotide identity (ANI), but vary in the presence of coronatine-associated genes., Pseudomonas syringae pv. tomato causes bacterial speck in tomato. We report the genome sequences of two P. syringae pv. tomato strains, J4 and J6, that are genetically closely related, with >99.9 average nucleotide identity (ANI), but vary in the presence of coronatine-associated genes.

Summary Bacterial spot caused by Xanthomonas perforans (Xp) is an economically important disease in tomato. Previous studies have shown that the recently isolated Xp strains have acquired and retained the effector gene, xopJ2, which has been reported to increase fitness of the pathogen in the field. To elucidate the fitness benefit of xopJ2, we quantified the effect of xopJ2 on the dispersal and evolution of Xp populations on tomato. We compared movement of two wild‐type Xp strains expressing xopJ2 to their respective xopJ2 mutants when co‐inoculated in the field. We developed a binary logistic model to predict the presence of Xp over spatial and temporal dimensions with or without xopJ2. Based on the model, wild‐type bacteria were dispersed approximately three times faster than the xopJ2 mutants. In a simulation experiment, the selective advantage due to increased dispersal velocity led to an increase in the frequency of xopJ2 gene in the Xp population and its apparent fixation within 10 to 12 cropping seasons of the tomato crop. Our results show that the presence of a single gene can affect the dispersal of a bacterial pathogen and significantly alter its population dynamics.

Before 1991

International audience; Multilocus sequence analysis for xanthomonads indicated a very close relationship between Xanthomonas gardneri, a tomato and pepper pathogen, and Xanthomonas cynarae, an artichoke pathogen. Whole-genome comparisons of representative strains from the two species revealed that the average nucleotide identity between the two species was above the threshold of 95-96%, and the two species could be merged into a single species. The type-III secretion systems of the two species were nearly identical, with minor differences in two genes. Furthermore, comparison of the type-III effector profiles showed high similarity. Given the close relationship between the species, we speculated that the two organisms might cross-infect. Inoculation of X. gardneri into artichoke resulted in a moderate disease reaction, although the disease on the bracts was very weak. Furthermore, following infiltration of leaves, X. cynarae reached significantly higher internal populations than X. gardneri. Infiltration of both species into pepper showed that the two species grew equally well and caused typical bacterial spot lesions. However, infiltration of X. cynarae at high concentration into tomato leaflets resulted in a hypersensitive reaction (HR). We have identified a unique gene associated with this HR and created a deletion mutant of this gene to determine whether this is the host-limiting factor. The mutant strain elicited an HR, indicating the presence of at least one additional factor in X. cynarae that limits its ability to colonize tomato.

An unusual fluorescent pseudomonad was isolated from tomato exhibiting leaf spot symptoms similar to bacterial speck. Strains were fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco and produced a soft rot on potato slices. However, the strains produced an unusual yellow, mucoid growth on media containing 5 % sucrose that is not typical of levan. Based on multilocus sequence analysis using 16S rRNA, gap1, gltA, gyrB and rpoD, these strains formed a distinct phylogenetic group in the genus Pseudomonas and were most closely related to Pseudomonas viridiflava within the Pseudomonas syringae complex. Whole-genome comparisons, using average nucleotide identity based on blast, of representative strain GEV388T and publicly available genomes representing the genus Pseudomonas revealed phylogroup 7 P. viridiflava strain UASW0038 and P. viridiflava type strain ICMP 2848T as the closest relatives with 86.59 and 86.56 % nucleotide identity, respectively. In silico DNA–DNA hybridization using the genome-to-genome distance calculation method estimated 31.1 % DNA relatedness between GEV388T and P. viridiflava ATCC 13223T, strongly suggesting the strains are representatives of different species. These results together with Biolog GEN III tests, fatty acid methyl ester profiles and phylogenetic analysis using 16S rRNA and multiple housekeeping gene sequences demonstrated that this group represents a novel species member of the genus Pseudomonas . The name Pseudomonas floridensis sp. nov. is proposed with GEV388T (=LMG 30013T=ATCC TSD-90T) as the type strain.

Recently, in Central Florida tomato production fields, tomato foliage and fruit were observed with symptoms similar to bacterial speck. Fluorescent pseudomonads were consistently isolated and the strains were characterized by standard LOPAT tests, pathogenicity tests, and genetic characterization using 16S ribosomal RNA (rRNA) sequences and multilocus sequence analysis (MLSA) of conserved housekeeping genes. LOPAT test results indicated that the strains were likely Pseudomonas cichorii. These strains were pathogenic on tomato and were also pathogenic on lettuce, the host for the type strain of P. cichorii. Likewise, strains of P. cichorii isolated in Florida since the early 1980s from hosts other than tomato, along with the type strain, were also pathogenic on tomato. Genetic characterization using 16S rRNA and MLSA confirmed that the strains were most closely related to P. cichorii but varied significantly from the type strain. The Florida P. cichorii strains formed a separate phylogenetic group along with P. cichorii strains isolated from tomato in Tanzania. These strains were different from the previously described morphotypes and genomovars of P. cichorii. Our results indicate the presence of a genetically distinct group of multihost pathogenic P. cichorii strains that have been present in Florida since at least the early 1980s.

Over a 2-year period, samples of tomato suspected of being infected with bacterial spot were collected across Indiana and sent to the Purdue Plant and Pest Diagnostic Laboratory (PPDL). A total of 50 strains were collected over 2 years. An effort was made to collect from both fresh market and processing tomato fields (22 from fresh market, 25 from processing, and 3 unspecified). The PPDL screened samples for bacterial spot, and they isolated and confirmed the pathogen. The Xanthomonas isolates were then sent to Jeff Jones at the University of Florida to determine taxonomic identification, race determination, and sensitivity to copper at 50 ppm and streptomycin at 200 ppm. The majority of Xanthomonas strains collected (78%) were classified as X. perforans. The majority of the X. perforans strains were designated race T4. A total of 12% of strains were classified as X. gardneri, all of which were collected north of U.S. I-70, a route that bisects the state into approximately even halves. X. euvesicatoria was only observed on one sample. X. vesicatoria was not observed. Of all strains collected in Indiana, 84% were copper tolerant. In contrast, only 58% of the strains were insensitive to streptomycin.

BackgroundGene transfer agents (GTAs) are phage-like mediators of gene transfer in bacterial species. Typically, strains of a bacteria species which have GTA shows more recombination than strains without GTAs. GTA-mediated gene transfer activity has been shown for few bacteria, with Rhodobacter capsulatus being the prototypical GTA. GTA have not been previously studied in plant pathogenic bacteria. A recent study inferring recombination in strains of the bacterial spot xanthomonads identified a Nigerian lineage which showed unusual recombination background. We initially set out to understand genomic drivers of recombination in this genome by focusing on mobile genetic elements.ResultsWe identified a unique cluster which was present in the Nigerian strain but absent in other sequenced strains of bacterial spot xanthomonads. The protein sequence of a gene within this cluster contained the GTA_TIM domain that is present in bacteria with GTA. We identified GTA clusters in other Xanthomonas species as well as species of Agrobacterium and Pantoea. Recombination analyses showed that generally, strains of Xanthomonas with GTA have more inferred recombination events than strains without GTA, which could lead to genome divergence.ConclusionThis study identified GTA clusters in species of the plant pathogen genera Xanthomonas, Agrobacterium and Pantoea which we have named XpGTA, AgGTA and PaGTA respectively. Our recombination analyses suggest that Xanthomonas strains with GTA generally have more inferred recombination events than strains without GTA. The study is important in understanding the drivers of evolution of bacterial plant pathogens.

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of two decades including a shift in race, diversification into three genetic groups, and host range expansion to pepper. Recombination originating from X. euvesicatoria was identified as the primary factor driving the diversification of X. perforans in Florida. The aim of this study was to genetically characterize X. perforans strains that were isolated from tomato and pepper plants grown in Alabama and compare them to the previously published genomes available from GenBank. Surprisingly, a maximum likelihood phylogeny coupled with a Bayesian analysis of population structure revealed the presence of two novel genetic groups in Alabama, which each harbored a different transcription activation-like effector (TALE). While one TALE, avrHah1, was associated with adaptation of X. perforans to pepper, the other was identified as a new class within the avrBs3 family, designated here as pthXp1. Examination of patterns of homologous recombination between X. perforans and other closely related Xanthomonas spp. indicated that the lineages identified here emerged in part through recent recombination events originating from xanthomonads associated with diverse hosts of isolation. Our results also suggest that the evolution of pathogenicity to pepper has likely emerged independently within X. perforans and in one lineage, was associated with the recombination-mediated remodeling of the Xps type II secretion and TonB transduction systems.ImportanceThe emergence of novel pathogen lineages has important implications in the sustainability of genetic resistance as a disease management tool in agricultural ecosystems. In this study, we identified two novel lineages of X. perforans in Alabama. While one lineage was isolated from symptomatic pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, pthXp1. Unlike AvrBs4, PthXp1overcomes Bs4-mediated resistance in tomato, indicating the evolution of this novel lineage towards fitness on this host. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts which results in the emergence of novel pathogen lineages and associated phenotypes, including host range expansion.

Outbreaks of bacterial spot on tomato (BST) caused by Xanthomonas perforans are a major concern for sustainable crop production. BST is a common occurrence in tomato transplants grown for field production. We hypothesized that BST outbreaks in commercial fields originate from X. perforans strains inadvertently introduced from commercial transplant facilities. To test this hypothesis, we used a genome-wide single-nucleotide polymorphism (SNP) analysis to characterize X. perforans strains recovered from tomato transplant facilities and fields in commercial production areas. X. perforans strains were isolated from symptomatic transplants prior to roguing at two commercial transplant growers. Then, the same groups of transplants were tracked to commercial fields to recover X. perforans strains from diseased plants prior to harvest. Whole-genome sequencing was carried out on 84 strains isolated from transplant and field plants from Florida and South Carolina. SNPs were called using three reference strains that represented the genetic variation of the sampled strains. Field strains showing genetic similarity to transplant strains had a difference of 2 to 210 SNPs. Transplant and field strains clustered together by grower within each phylogenomic group, consistent with expectations. The range of genetic divergence among strains isolated from field plants was similar to the range obtained from strains on transplants. Using the range of genetic variation observed in transplants, we estimate that 60% to 100% of field strains were an extension of the transplant strain population. Our results stress the importance of BST management to reduce X. perforans movement from transplant to field and to minimize subsequent disease outbreaks. IMPORTANCE Current management of Xanthomonas perforans on tomato plants mainly relies on the frequent application of pesticides. However, the lack of effective pesticides and the development of strain tolerance to certain bactericides limit the ability to control outbreaks in production fields. Better knowledge of probable sources of X. perforans inoculum during tomato production is required to refine management strategies. Tomato plants are typically established in the field using transplants. This study aimed to determine if strains from field epidemics were coming from transplant facilities or resulted from local field outbreaks. The overall goal was to identify potential sources of inoculum and subsequently develop strategies to reduce carryover from transplant production to the field. Our results indicate that tomato producers should shift disease management efforts to transplant facilities to reduce disease in the field. Improved transplant health should reduce the likelihood of bacterial spot outbreaks and subsequently reduce pesticide usage in the field.

Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be “core” genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida.

Xanthomonas axonopodis pv. vesicatoria, causal agent of bacterial spot of tomato (Lycopersicon esculentum L.) and sweet pepper (Capsicum annuum L.) was isolated from tomato and pepper plants in greenhouse production in the Province of Antalya, in southwestern Turkey. Disease incidence was less than 4% of plants observed in 2001 and ranged from 1 to 20% in 2002. Eleven seedling-producing companies and 26 greenhouses that produce tomato and pepper were surveyed during the rainy seasons of 2001 and 2002. The increase in disease incidence in 2002 is an indication that this disease is becoming more prevalent on tomato and pepper plants grown in greenhouses in southwestern Turkey. A gram-negative bacterium producing yellow-pigmented colonies on nutrient agar was consistently isolated from brown, circular spots on leaflets of tomato and sweet pepper seedlings. Five isolates were pathogenic on commercial cultivars of tomato and pepper when bacterial suspensions (108 CFU/ml) were infiltrated into the intercellular spaces of leaves to determine race by using procedures described by Bouzar et al. (1). All the isolates produced hypersensitive reaction responses on tomato genotype cv. Hawaii 7998 and pepper genotype cvs. 20 R and 30 R and were designated tomato race 1 pepper race 1 (T1P1) (1). Fatty acid analysis of the strains identified them as X. axonopodis vesicatoria with similarity index values of 0.872 to 0.933. In addition, the strains were tested with X. axonopodis vesicatoria-specific polymerase chain reaction primers (RST 2/3 and RST 9/10) (2). The isolates were determined to be X. axonopodis vesicatoria. Although bacterial spot of tomato has been suspected in Turkey for a number of years, to our knowledge, this is the first report of the bacterium on tomato. References: (1) H. Bouzar et al. Phytopathology 84:663, 1994. (2) R. P. Leite, Jr. et al. Appl. Env. Microbiol. 60:1068, 1994.

Bacterial spot (BS) has been reported as an important disease on pepper in Nigeria (4). Xanthomonas campestris pv. vesicatoria was identified as the causal agent using phenotypic and pathogenicity tests; however, X. campestris pv. vesicatoria is a synonym for two genetically distinct groups that have been elevated to the species X. euvesicatoria and X. vesicatoria (2). Furthermore, the latter two species and X. gardneri cause similar diseases on pepper (2). In order to determine the species associated with BS on pepper, leaves with irregular, dark brown lesions were collected from pepper plants in fields from northwestern Nigeria, and isolations were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated. Six strains isolated from pepper were determined to be non-amylolytic. For race determinations, bacterial suspensions of the pepper strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The six pepper strains elicited HRs on the tomato differential genotypes. The strains produced a susceptible reaction on all pepper differentials and were designated as pepper race 6 (5). Multilocus sequence analysis (MLSA) using six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze the strains (1) (GenBank Accession Nos. KJ938585 to KJ938587, KJ938592 to KJ938594, KJ938599 to KJ938601, KJ938606 to KJ938608, KJ938633 to KJ938635, and KJ938640 to KJ938642). A partial sequence of hrpB2 was also sequenced since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence (3) (KJ938622 to KJ938627). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the NCBI database using muscle tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the Nigerian pepper strains were identical to the X. euvesicatoria reference strain 85-10. Although BS is common in Nigeria, to our knowledge, this represents the first report for this pepper pathogen in Nigeria. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (3) J. B. Jones et al. System Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (2) E. U. Opara and F. J. Odibo. J. Mol. Gen. 1:35, 2009. (5) R. E. Stall et al. Ann. Rev. Phytopathol. 47:265, 2009.

Bacterial spot (BS) is an important disease of tomato in Nigeria (2). Although a xanthomonad was isolated from tomato in Nigeria and characterized using phenotypic and pathogenicity tests, the bacterium was not characterized genetically to confirm the species. To determine the species associated with BS, leaves were collected in fields in northwestern Nigeria from tomato plants showing typical BS symptoms, which consisted of dark, irregular-shaped brown leaf spots that coalesced, resulting in a blighted appearance. Isolations from individual lesions were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated from 14 lesions and all were determined to be amylolytic (3). To determine the races of these strains, bacterial suspensions of the tomato strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The tomato strains elicited hypersensitive reactions (HRs) on the four pepper differential lines and an HR on the tomato genotype FL 216, which contains the R gene Xv3, but elicited susceptible reactions on the tomato genotypes Hawaii 7998 and Bonny Best. These reactions are typical of X. perforans tomato race 3 strains (5). Multilocus sequence analysis (MLSA) of six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze four representative strains (1) (GenBank Accession Nos. KJ938581 to KJ938584, KJ938588 to KJ938591, KJ938595 to KJ938598, KJ938602 to KJ938605, KJ938629 to KJ938632, and KJ938636 to KJ938639, respectively). A partial sequence of hrpB2 was also made since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence of this gene (3) (KJ938609 to KJ938621 and KJ938628). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the National Center for Biotechnology Information (NCBI) database ( www.ncbi.nlm.nih.gov ) using the MUSCLE tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the strains grouped most closely with the X. euvesicatoria reference strain 85-10 but more distantly to X. perforans. The hrpB2 sequence, which is highly conserved for each Xanthomonas species pathogenic on tomato (4), was sequenced from the tomato strains. These sequences were identical to the hrpB2 sequence from X. perforans strains but different from X. euvesicatoria. Although BS is common in Nigeria, to our knowledge, this represents a unique group of X. euvesicatoria strains from tomato that are identical to X. perforans based on pathogenic reactions on tomato and pepper and hrpB2 sequence identity but are more closely related to X. euvesicatoria based on the six housekeeping gene sequences. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) E. U. Opara and F. J. Odibo. J. Mol. Genet. 1:35, 2009. (3) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (5) R. E. Stall et al. Annu. Rev. Phytopathol. 47:265, 2009.

International audience; Multilocus sequence analysis of Xanthomonas species revealed a very close relationship between Xanthomonas cynarae , an artichoke pathogen and Xanthomonas gardneri, a tomato and pepper pathogen. Results of whole genome sequence comparisons using average nucleotide identity between representative strains of X. gardneri and X. cynarae were well above the threshold of 95–96 %. Inoculations of X. gardneri strains in artichoke leaves caused mild disease symptoms, but only weak symptoms were observed in the bracts. Both X. cynarae and X. gardneri grew equally and caused typical bacterial spot symptoms in pepper after artificial inoculation. However, X. cynarae induced a hypersensitive reaction in tomato, while X. gardneri strains were virulent. Pathogenicity-associated gene clusters, including the protein secretion systems, type III effector profiles, and lipopolysaccharide cluster were nearly identical between the two species. Based on our results from whole genome sequence comparison, X. gardneri and X. cynarae belong to the same species. The name X. cynarae has priority and X. gardneri should be considered as a later heterotypic synonym. An emended description of X. cynarae (type strain=CFBP 4188T, =DSM 16794T) is given. However, due to the host specificity in artichoke and tomato, two pathovars, X. cynarae pv. cynarae and X. cynarae pv. gardneri, are proposed.

Bacterial spot disease caused by Xanthomonas euvesicatoria is one of the major constraints for pepper production in Florida and worldwide. The common prevalence of copper (Cu)-tolerant X. euvesicatoria strains has reduced the efficacy of Cu based bactericides against bacterial spot of pepper. In this study, we evaluated antimicrobial activity of three Cu-based nanocomposites, namely, core-shell Cu (CS–Cu), fixed-quat Cu (FQ-Cu) and mixed-valence Cu (MV-Cu), along with Kocide® 3000, which is a micron size metallic Cu bactericide and is commercially available, both in vitro and in greenhouse trials, against a Cu-tolerant strain of X. euvesicatoria. In repeated in vitro tests, X. euvesicatoria growth was completely inhibited 24 h after exposure of bacterial cells to 500 and 1000 μg/ml of MV-Cu and all concentrations of FQ-Cu and CS-Cu (100, 200, 500, and 1000 μg/ml) treated for 1 h. Micron size copper, even at 1000 μg/ml, had no statistical difference in growth in comparison to the untreated control (P = 0.05; LSD). In the repeated greenhouse experiments, plants treated with all the copper composites exhibited less bacterial spot severity than untreated control and similar or significantly less than Cu-mancozeb (P

Bacterial disease management is a challenge for modern agriculture due to rapid changes in pathogen populations. Genome sequences for hosts and pathogens provide detailed information that facilitates effector-based breeding strategies. Tomato genotypes have gene-for-gene resistance to the bacterial spot pathogen Xanthomonas perforans. The bacterial spot populations in Florida shifted from tomato race 3 to 4, such that the corresponding tomato resistance gene no longer recognizes the effector protein AvrXv3. Genome sequencing showed variation in effector profiles among race 4 strains collected in 2006 and 2012 and compared with a race 3 strain collected in 1991. We examined variation in putative targets of resistance among Florida strains of X. perforans collected from 1991 to 2006. Consistent with race change, avrXv3 was present in race 3 strains but nonfunctional in race 4 strains due to multiple independent mutations. Effectors xopJ4 and avrBs2 were unchanged in all strains. The effector avrBsT was absent in race 3 strains collected in the 1990s but present in race 3 strains collected in 2006 and nearly all race 4 strains. These changes in effector profiles suggest that xopJ4 and avrBsT are currently the best targets for resistance breeding against bacterial spot in tomato.

Whole genome sequencing of a copper resistant (CuR) black rot strain of Xanthomonas campestris pv. campestris (Xcc) isolated from a broccoli plant in Trinidad revealed a unique operon for copper resistance. The cop genes of strain Xcc-BrA1 were determined to be present on a 160 to 180 kb plasmid shown to be non-conjugative with other xanthomonads. While nucleotide comparison of a putative 8.0 Kbp copLABMGF gene cluster identified in Xcc-BrA1 genome did not reveal any homologous region with other known CuR Xanthomonas strains from diverse origins, the comparison of the translated amino acid sequence indicated similarity with X. citri, X. c. pv. citrumelonis and X. vesicatoria Cop proteins. Cloning of the copLAB gene cluster from Xcc-BrA1 conferred copper resistance to other copper-sensitive xanthomonads. Although Xcc-BrA1 harbors copLAB genes with similar sizes and organization and is able to grow on Cu-amended medium as other CuR xanthomonads, the phylogenetic analysis of nucleotide sequences indicates that the cop cluster in Xcc-BrA1 is unique and distantly related to other copLAB genes from Xanthomonas and Stenotrophomonas. The origin of copper resistance genes in Xcc-BrA1 is likely a result of horizontal gene acquisition from a still unknown phylloplane cohabitant. The findings of this study have implications for the management of crop diseases caused by CuR xanthomonads. Future studies could focus on and determining the distribution, overall importance and appropriate control measures for strains harbouring these unique genes.

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ), and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs314EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs314EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin‐binding domain characteristic amino acid sequence motif (GPLL) and a C‐terminal domain‐containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two‐hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2‐SPLL and AvrGf2‐AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin–AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin‐binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C‐terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site‐directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.

Citrus surveys were conducted at high (>700 m), medium (300-600 m) and low (

Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three (ORFA, ORF2 and ORF4) were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases.

Recombination is a major driver of evolution in bacterial populations, because it can spread and combine independently evolved beneficial mutations. Recombinant lineages of bacterial pathogens of plants are typically associated with the colonization of novel hosts and the emergence of new diseases. Here we show that recombination between evolutionarily and phenotypically distinct plant-pathogenic lineages generated recombinant lineages with unique combinations of pathogenicity and virulence factors. Xanthomonas euvesicatoria and Xanthomonas perforans are two closely related lineages causing bacterial spot disease on tomato and pepper worldwide. We sequenced the genomes of atypical strains collected from tomato in Nigeria and observed recombination in the type III secretion system and effector genes, which showed alleles from both X. euvesicatoria and X. perforans . Wider horizontal gene transfer was indicated by the fact that the lipopolysaccharide cluster of one strain was most similar to that of a distantly related Xanthomonas pathogen of barley. This strain and others have experienced extensive genomewide homologous recombination, and both species exhibited dynamic open pangenomes. Variation in effector gene repertoires within and between species must be taken into consideration when one is breeding tomatoes for disease resistance. Resistance breeding strategies that target specific effectors must consider possibly dramatic variation in bacterial spot populations across global production regions, as illustrated by the recombinant strains observed here. IMPORTANCE The pathogens that cause bacterial spot of tomato and pepper are extensively studied models of plant-microbe interactions and cause problematic disease worldwide. Atypical bacterial spot strains collected from tomato in Nigeria, and other strains from Italy, India, and Florida, showed evidence of genomewide recombination that generated genetically distinct pathogenic lineages. The strains from Nigeria and Italy were found to have a mix of type III secretion system genes from X. perforans and X. euvesicatoria , as well as effectors from Xanthomonas gardneri . These genes and effectors are important in the establishment of disease, and effectors are common targets of resistance breeding. Our findings point to global diversity in the genomes of bacterial spot pathogens, which is likely to affect the host-pathogen interaction and influence management decisions.

Type III secretion system effectors contribute to pathogenicity through various mechanisms. Recent surveys showed an increasing prevalence of the type III secretion effector avrBsT among Xanthomonas perforans strains. We hypothesized that the acquisition of avrBsT has a fitness advantage for the pathogen. The contribution of avrBsT to fitness on tomato was evaluated based on disease severity, in planta growth, competition, and recovery rates of wild-type (WT) and avrBsT mutant strains in greenhouse and field plants. GEV872 and GEV1001, representative strains of two phylogenomic groups of X. perforans, were selected for generating avrBsT mutants. Disease severity was higher for WT strains compared with the avrBsT mutant strains. X. perforans WT and avrBsT mutant strains did not differ following leaf infiltration of greenhouse plants in direct competition and in planta growth assays. The effect of avrBsT on pathogen fitness was noticeable under field conditions. Differences in strain recovery were significant, with WT being recovered two to eight times more than avrBsT mutant strains in the case of both strains GEV872 and GEV1001. WT strains were capable of spreading longer distances across field plots compared with avrBsT mutant strains. Findings suggest that the functional AvrBsT affects the fitness of X. perforans under field conditions, making it an ideal candidate for bacterial spot resistance breeding efforts in tomato.

Background: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. Results: We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. Conclusions: We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods. EEA Bella Vista Fil: Gochez, Alberto Martin. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina Fil: Huguet-Tapia, Jose Carlos. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Minsavage, Gerald V. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Shantharaj, Deepak. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Jalan, Neha. University of Florida. Department of Microbiology and Cell Science. Citrus Research and Education Center; Estados Unidos Fil: Strauß, Annett. University of Tübingen; Alemania Fil: Lahaye, Thomas. University of Tübingen; Alemania Fil: Wang, Nian. University of Florida. Department of Microbiology and Cell Science. Citrus Research and Education Center; Estados Unidos Fil: Canteros, Blanca Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina Fil: Jones, Jeffrey B. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Potnis, Neha. Auburn University. Department of Entomology and Plant Pathology; Estados Unidos

Xanthomonas citri pv. citri strain 306 ( Xcc 306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc 306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α- l -arabinofuranosidase, and the xyn43F gene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4- O -methylglucuronoxylan (MeGX n ) to oligoxylosides methylglucuronoxylotriose (MeGX 3 ), xylotriose (X 3 ), and xylobiose (X 2 ). Xcc 306 completely utilizes MeGX n predigested with Xyn10A or Xyn10C but shows little utilization of MeGX n . Xcc 306 with a deletion in the gene encoding α-glucuronidase ( Xcc 306 Δ agu67 ) will not utilize MeGX 3 for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGX n . Preferential growth on oligoxylosides compared to growth on polymeric MeGX n indicates that GH10 xylanases, either secreted by Xcc 306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10 , agu67 , cirA , the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc 306 GH10 xylanases on MeGX n . The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc 306 and, ultimately, to plant disease.

Citrus canker is caused by two Xanthomonas species, Xanthomonas citri, which has become the primary pathogen where citrus canker occurs (type A citrus canker, Xc-A), and X. fuscans pv. aurantifolii (Xfa), which consists of strains B and C. The B strain is less pathogenic than the A strain, but produces symptoms in all citrus species. The C-type cankers only infect Key lime (Citrus aurantifolia) and produce a hypersensitive reaction (HR) in grapefruit (Citrus paradisi) leaves. An avirulence gene, avrGf2, was identified in a C strain that was responsible for the HR in grapefruit. AvrGf2 is a member of XopAG effector family and shares 45% identity at amino acid level with another member of the same family, AvrGf1 from strain Xc-Aw, which was previously shown to elicit an HR in grapefruit. AvrGf2 shares sequence identity with other XopAG effectors present in Xanthomonas vasculorum, Xanthomonas campestris pv. musacearum and Pseudomonas syringae pv. tomato. Mutagenesis of avrGf2 in C strain resulted in a compatible reaction in grapefruit. There was no observable effect on virulence when Xc-A transconjugants containing either avirulence gene were inoculated on Key lime. Expression of avrGf1 or avrGf2 in Xc-A resulted in a similar phenotype following infiltration into grapefruit leaves, although the avrGf2 transconjugant elicited a faster HR and lower populations than the transconjugant containing avrGf1. Also, it was shown that all Xfa-B strains tested contain a transposon in avrGf2 that helps to explain the differences in host range between B and C strains.